Site‐Specific Multi‐Functionalization of the Carrier Protein CRM197 by Disulfide Rebridging for Conjugate Vaccine Development

Abstract Conjugation of an antigen to a carrier protein is widely used for vaccine development. To develop the next generation of conjugate vaccines, we describe here a method for the controlled multi‐functionalization of the widely employed carrier protein CRM197 with a carbohydrate‐based antigen and an immune potentiator. The approach is based on the selective reduction of one of the disulfides of CRM197 followed by disulfide rebridging employing an appropriately functionalized dibromopyridazinedione. Efficient protein modification required that the reduction and functionalization with a dibromopyridazinedione was performed as a one‐step procedure with control over the reaction temperature. Furthermore, ligations were most successful when dibromopyridazinediones were employed having a functional entity such as a TLR7/8 agonist and a cyclooctyne for further modification. Site‐specific conjugation avoids modification of T‐epitopes of the carrier protein and covalent attachment of an immune potentiator will ensure that cytokines are produced where the vaccine interacts with relevant immune cells resulting in efficient immune potentiation.


General methods
All purchased chemicals were used without further purification unless stated otherwise. CRM197 was purchased from Scarab Genomics. Solvents were dried over activated 4 Å molecular sieves. Aqueous solutions of salts were saturated unless stated otherwise. Concentration of organic solutions was performed under reduced pressure at 40 °C. Thin layer chromatography (TLC) was performed on silica gel 60F254 with detection by UV light (254 nm) and staining by p-anisaldehyde solution, followed by heating for visualization. Column chromatography was performed on silica gel G60 (0.040 -0.063 mm). Analytical HPLC was performed on a Shimadzu HPLC system coupled to a ELSD detector (Alltech3300) using ZIC-HILIC (SeQuant, 5 µm), C5 (Phenomenex Jupiter, 5 µm) or a C18 column (Zorbax Eclipse XDB, 5 µm). Preparative HPLC was performed on a Puriflash4125 using HILIC, C4 or C18 columns. NMR spectra were recorded on a Bruker Avance III 600 instrument (600.22 MHz for 1 H, 150.93 MHz for 13 C) using standard Bruker NMR software. Chemical shifts are reported in parts per million (ppm). NMR data is presented as: chemical shift, multiplicity (where s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet), the coupling constant in Hertz (Hz), and allocation. Assignments were based on COSY, HSQC and HMBC data. HRMS data were obtained on a Micromass Q-TOF Ultima Global instrument. Protein mass determination was performed on an Agilent 6560 drift tube IMS (DTIMS)-quadrupole time-off-flight (Q-TOF) MS instrument using a UPLC column (Waters Bioresolve -RP.mAb, 2.7 µm, 2.1×50 mm). MALDI-TOF measurements were performed on a Bruker Autoflex and concentration of Proteins was determined with a DeNovix Nanodrop spectrometer, using for CRM197-derivatives an E1 % value of 10.7.

Automated solid-phase peptide synthesis
The azide equipped hexapeptide was synthesized via Fmoc-solid-phase Peptide Synthesis (SPPS) with a microwave-assisted peptide synthesizer (CEM HT12 Liberty Blue). Rink Amide AM resin (25 µmol) was swollen in 10 mL of a 1:1 mixture of DMF/DCM for 5 min, drained, and then treated with piperidine (20 % in DMF; 10 mL) for 65 seconds at 90 °C, followed by draining, and washing with DMF (3 × 5 mL). The resin was then treated with the Fmoc protected Amino acid (0.2 mol/L, 0.625 mL, 5 eq), DIC ( 1 mol/L, 0.25 mL, 10 eq) and Oxyma Pure (1 mol/L, 0.125 mL, 5 eq) in DMF (4 mL) at 76 °C for 15 s before the temperature was increased to 90 °C for an additional 110 s following by draining of the solvent. Fmoc was cleaved as described above and the resin was treated with the next amino acid. After full assembly of the hexapeptide, a small amount of the resin was treated with 1 drop of TFA in MeCN/water (1/1), it was stirred for 1 h and MALDI-TOF analysis of the supernatant revealed full conversion to the desired peptide.

Manual assembly of the TLR1/2 agonist
Commercially available Pam2Cys (28 mg; 31 µmol) was dissolved in dry DMF (1 mL) followed by the addition of Oxyma Pure (4.4 mg; 31 µmol) and DIC (7.2 µL; 47 µmol) and it was stirred for 3 min at 22 °C. Subsequently, the resin bound peptide (ca. 16 µmol) was added and it was stirred for 16 h more.
Then, the resin was filtered, washed with DMF (5 times) and stirred in 20 % piperidine (1 mL
The organic phase was dried over Na2SO4, the solvent was removed in vacuo and the crude product was purified with silica gel chromatography (DCM/MeOH = 20/0 → 20/1) to obtain product 19 (

Installation of linker 1 on CRM197
For assessment of best reaction conditions, the linker introduction was screened with two different methods: Method 1: CRM197 (100 µg; 1.7 nmol) was dissolved in 100 µL of buffer, followed by the addition of a solution of TCEP × HCl (5 µL, 3.5 mM in water) and is was stirred at 37 °C for 3 h. Then, the solution was spin filtered (cut-off 10 kDa, 3 times against fresh buffer) to a final Volume of 100 µL, linker 1 (2 µL; 17 mM in DMSO) was added and stirring was continued for the indicated time. The reaction mixture was spin filtered against water as described above and was then analyzed by ESI-QTOF-MS.
Method 2: CRM197 (100 µg; 1.7 nmol) was dissolved in 100 µL of buffer, followed by the addition of linker 1 (2 µL; 17 mM in DMSO) and after stirring for 5 min a solution of TCEP × HCl (5 µL, 3.5 mM in water; 10 eq.) was added. It was stirred at the indicated temperature and time and then the sample was spin filtered against water (cut-off 10 kDa, 3 times), followed by analysis of the reaction mixture by ESI-QTOF-MS.  Optimized procedure: CRM197 (2 mg; 34.2 nmol) was dissolved in TRIS-buffer (2 mL; 250 mM, pH = 8), followed by the addition of the linker (40 µL; 10 mg/mL in DMSO; 684.9 nmol) and after stirring for 5 min it was cooled down to 4 °C. Then, a solution of TCEP × HCl (50 µL, 2 mg/mL in water; 342.5 nmol) was added and stirring was continued for 40 h at 0 °C. After that, the sample was spin filtered against water (cut-off 10 kDa, 3 times) which afforded 380 µL aqu. solution of 2. Nanodrop analysis (E 1% = 10.7) revealed a concentration of 5 mg/mL which refers to a total protein recovery of 1.9 mg (95 %) and analysis by ESI-QTOF-MS revealed product purity of ˃ 95 %

Synthesis of conjugate 8
To a solution of linker conjugated CRM 2 (12 µL; 8.4 mg/mL in water; 1.69 nM) was added 90 µL PBSbuffer (pH = 7.4). Then, a solution of 3 (1.5 µL; 5 mg/mL in water; 16.9 nM) was added and it was stirred at room temperature for 16 h. After that, the sample was spin filtered against water (cut-off 10 kDa, 3 times) and analysed by ESI-QTOF-MS which revealed full conversion to the desired product. Figure 4 ESI-QTOF spectra of 8

Synthesis of conjugate 9
To a solution of linker conjugated CRM 2 (10 µL; 5 mg/mL in water; 0.85 nM) was added 40 µL PBSbuffer (pH = 7.4). Subsequently, a solution of 4 (2 µL; 10 mg/mL in water; 16.9 nM) was added and it was stirred at 37 °C for 16 h. After that, the sample was spin filtered against water (cut-off 10 kDa, 3 times) which afforded 90 µL aqu. solution. Nanodrop analysis (E 1% = 10.7) revealed a concentration of 0.39 mg/mL which refers to a total protein recovery of 35 µg (70 %) and analysis by ESI-QTOF-MS revealed full conversion to the desired product.

Synthesis of conjugate 23
To a solution of linker conjugated CRM 22 (27 µL; 1.8 mg/mL in water; 0.85 nM) was added 27 µL PBSbuffer (pH = 7.4). Subsequently, a solution of 4 (2 µL; 10 mg/mL in water; 16.9 nM) was added and it was stirred at 37 °C for 16 h. After that, the sample was spin filtered against water (cut-off 10 kDa, 3 times) which afforded 100 µL aqu. solution. Nanodrop analysis (E 1% = 10.7) revealed a concentration of 0.37 mg/mL which refers to a total protein recovery of 37 µg (74 %) and analysis by ESI-QTOF-MS indicated product purity of ˃ 80 %

Random conjugation
Glycan 28 (0.38 mg; 0.51 µmol) and Bis NHS ester of adipinic acid [5] (1.7 mg; 5.1 µmol) were dissolved in DMSO (0.3 mL) followed by the addition of NEt3 (0.29 µL; 2.0 µmol) and the mixture was stirred for 4 h at 22 °C. Then, EtOAc (0.6 mL) and PBS buffer (0.3 mL; pH = 7.4) were added and after vigorous shaking the organic phase was removed with a pipette. The aqueous phase was washed two more times with EtOAc and residues of the organic solvent were then removed by bubbling air through the solution. The remaining solution was divided in three portions of which each was either added to a solution of CRM197 (0.2 mg in 0.1 mL water; 3.4 nM) or CRM-derivative 22 (0.2 mg in 0.1 mL water; 3.4 nM). The conjugation reaction were gently shook at 22 °C for 16 h and were then purified with spin filtration against water (cut-off 10 kDa, 3 times). Analysis of the conjugates with MALDI-TOF revealed a distribution of protein glycan/carrier: 7.4 (CRM197 based conjugate) and 6.9 (22 based conjugate).
were removed, spin filtered three times against water (cut-off 10 kDa) and analyzed by ESI-TOF MS.
The remaining reaction mixture was incubated for 19 h more, before it was treated and analyzed in as described above.

SI Figure 24
ESI-QTOF MS of pure conjugate 22 and treated with 5 µM GSH after 5 and 24 h Conditions B: To conjugate 22 (70 µg in 60 µL water) was added GSH in PBS buffer (340 µL, 5 µL GSH, pH = 6.5) and the reaction mixture was incubated for 5 h at 37 °C. Then, 200 µL of the reaction mixture were removed, spin filtered three times against water (cut-off 10 kDa) and analyzed by ESI-QTOF MS.
The remaining reaction mixture was incubated for 19 h more but was not analyzed further, since full linker cleavage was already detected after 5 h. ESI-QTOF MS of conjugate 22 treated with 5 µM GSH after 5 and 24 h